Follicle Stimulating Hormone
About Follicle Stimulating Hormone (FSH) is a protein hormone in the gonadotropin class. Gonadotropins stimulate the reproductive structures in both males and females, and are secreted by cells called gonadotrophs in the anterior pituitary gland. As the name implies, FSH stimulates the maturation of the ovarian follicles in females. When given pharmaceutical FSH, subjects expressed "superovulation," or development of more than the normal number of follicles, and therefore experienced an increased number of mature gametes. In males, FSH has also proved to be critical in the process of sperm production. The hormone supports the function of Sertoli cells which function to nourish the sperm during maturation. FSH Signaling FSH performs it's function through a series of specific signaling mechanisms. FSH is a disulfide-rich heterodimer which shares a common alpha subunit with other gonadotropins, however has a specific beta subunit which imparts hormone specificity. A signal is transmitted by FSH through the 75kDa receptor, which is composed 675 amino acids. The G-coupled protein receptor spans the cellular membrane seven times, with seven conserved alpha helixes. The crystal structure of FSH bound to the binding domain of the FSH receptor shows an interaction that resembles a handclasp. Ten parallel beta strands located on the receptor come into contact with the C terminal beta strands on the FSH protein. The receptor warps around the middle section of the hormone and interacts with the C terminal segments of other loops of both the FSH alpha and beta subunits. This interaction with FSH and its specific receptor causes the hormone to undergo a series of conformational adjustments, ultimately adopting the rigid structure required for protein signaling. As a result of this conformational change,the seven membrane-spanning domains that illicit guanine nucleotide, exchange in associated G coupled proteins, allowing a signal to undergo transduction. Through the ability of the FSH protein to adopt a confirmation in response to physical interactions with its specific receptor, the protein is successful in regulating certain aspects of the human reproduction system. DNA Construct used in Expression The FSH protein is 35.5kD glycoprotein dimer. As its structure is similar to that of other gonadotropins, it contains to polypeptide units labeled alpha and beta. The beta subunit consists of 111 amino acids, which allows for its specific biological interaction with the FSH-receptor. The gene for the FSH beta subunit is located on chromosome 11p13, and is expressed in gonadotrophs of the pituitary cells. Purification and Isolation of FSH In order to purify and isolate FSH and use it in a genetically engineered setting, the chromosomal DNA encoding this protein must first be isolated. This is done by fragmenting the DNA of chromosome 11, and attaching the fragments to a vector such as a bacteriophage. The bacteriophage is then taken up by competent Ecoli bacteria cells, where the DNA is amplified to establish a permanent library of cloned fragments. Once the cloned DNA has been confirmed by DNA sequencing, it is placed into mammalian cell vectors designed for high expression of the subunit mRNAs. Once the vectors are within the mammalian cell, the gene for the protein hormone can be replicated using host cell replication machinery. Since many factors such as steroids, and gonadotrohin releasing hormone, regulate the transcription of gonadotrophin RNA, each subunit gene is incorporated into a separate suitably designed expression vector which also contains separate amplifiers. The amplifiers are genes whose expression rates can be controlled by altering external genes. In some instances for example, the alpha subunit gene was linked to dihydofolate reductase, which is required for the synthesis of nucleic acid precursors. Subjecting cells to stimulants such as MTX causes amplification of the DHFR gene which in return will amplify the associated DNA. Commercial Production of hFSH The production of large scale FSH protein is done at a commercial level involving two major steps. First, a CHO cell culture process is performed, which is based on the large scale culture of the production clone in a bio-reactor. The working cell bank vials containing mammalian cells transfected with genes encoding the alpha and beta subunits are are grown in a bio-reactor. The bio-reactor is equipped with specific environmental control capabilities. The growth medium for the cells is continuously refreshed as cells are multiplied until sufficient numbers are achieved. The cells are then mixed with a suspension of micro carrier beads, to which the cells attach. After a defined period, and once the cells are numerous enough, the medium is changed to a medium that is suitable for the production of FSH, containing the necessary stimulants and co factors. The FSH production phase then follows in which conditioned medium is collected for downstream purification. Once the FSH is purified through immunoaffinity and chromatographic steps, it can be extracted from the supernatant, and utilized in a pharmaceutical sense. References 1."Follicle-stimulating Hormone." Wikipedia. Wikimedia Foundation, 21 Sept. 2014. Web. 21 Sept. 2014.http://en.wikipedia.org/wiki/Follicle-stimulating_hormone 2.Bowen, R. "Luteinizing and Follicle Stimulating Hormones." Luteinizing and Follicle Stimulating Hormones. N.p., n.d. Web. 21 Sept. 2014 http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/hypopit/lhfsh.html 3.Follicle Stimulating Hormone, Beta Polypeptide." Gene & Protein Summary: FSHB. European Bioinformatics Institue, n.d. Web. 21 Sept. 2014. http://www.ebi.ac.uk/s4/summary/molecular?term=FSHB&classification=9606&tid=nameOrgENSG00000131808 4.Walker, William H. "FSH and Testosterone Signaling in Sertoli Cells." Society of Reproduction and Fertility, n.d. Web. 21 Sept. 2014.http://www.reproduction-online.org/content/130/1/15.full 5.Howles, C. "Genetic Engineering of Human FSH (Gonal-F®)." Human Reproduction Update 2.2 (1996): 172-91. Web.